The comparison of ISSR and RAPD markers with

different species of

Triticum

M. Ebadi

* and Mahsa Eghbali

Assistant Professor, Damgan Branch, Islamic Azad University, Iran

Master of Genetic, Damgan Branch, Islamic Azad University, Iran

ABSTRACT

Considering genetic variety in Iran, we applied markers to determine genetic variety and phylogenic connections in

different species. On the other hand, we made an attempt to compare RADP and ISSR molecular markers on different

species of wheat considering the food consumption of Iran and easy sample to wheat. In this experimental study,

markers were applied to consider different species of wheat and they were experimented by RADP and ISSR starters.

The effect of marker was evaluated on different species of wheat by PCR test. A comparative effect of RADP and ISSR

markers via PCR method signi ed that different species of wheat based on applied starters with a clear distance from

DNA bands showed their similarities. In conclusion, the study results indicated to the effectiveness of different spe-

cies of wheat on markers. Low cost and availability to wheat species and applying their DNA can be more effective

in comparison to RADP and ISSR.

288

ARTICLE INFORMATION:

*Corresponding Author:

Received 27

Dec, 2016

Accepted after revision 2

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Biosci. Biotech. Res. Comm. Special Issue No 1:288-292 (2017)

INTRODUCTION

Although from too many years ago human had consid-

ered drug crops of plants, the increasing production of

these crops in  elds and gardens turned to a new sci-

ence since second half of the 20

century. Destroying

and consuming growing plants of nature were placed

by enjoying nature plants. Nowadays, the applying of

genetic engineering methods and biotechnology allow

forming DNA molecules with inheritance properties,

and a signi cant progression has created in biological

sciences. Modern biotechnology, a novel technology, is

able to increase the function of plants through changing

the genetic structure of crops and other plants.

Wheat (in science: Triticum) is a cereal grain. There

are wild and domestic types of wheat. It is belonged to

annual monocots magnoliophyta plants and Poaceae

and a member of Gramineae family.

Wheat is among the oldest crop plants used by human,

it is cultivated and harvested in a very great extent. Bot-

any, it is a member of Triticum species divided in three

different groups with determined chromosomes carrying

all genetic properties of the family (3).Two diploids are

more dif cult than other crops, as some of them belong

M. Ebadi and Mahsa Eghbali

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE COMPARISON OF ISSR AND RAPD MARKERS WITH DIFFERENT SPECIES OF

TRITICUM

289

to the wheat species of genetics with four polyploidy

chromosome chains, while other species got 6 chromo-

some chains. Diploids grow only in jungles and deserts

and cropped Einkorn wheat belong to this group.Wild

Tetraploids, and cultivated Emer and Durum belong to

this group. Hexaploids involves bread wheat 90000 hec-

tares of all 2.3 million hectares of water wheat are culti-

vated in cold regions.

PROPERTIES

Wheat contains all mineral salts. If you need to have dif-

ferent vitamins, take wheat. It is suggested to use wheat

gruel with sugar and almond to stop breast bleeding.

Wheat avoids gastric cancer.

RAPD MARKER

RADP marker starting a short oligonucleotid starter

propagates in PCR reaction with low annealing tem-

perature, a range of pieces of DNA pattern. One or more

piece of polymorph productions are created as a result of

changing in one open door in primer connection place

,and this polymorphism can be a genetic map (Kaiser

and Polatski, 1995) RADP marker is distributed in the

whole genome (Luine, Santanglo, 1994).

THE ADVANTAGES OF RAPD MARKER

It does not need the primary information on DNA orders

to design and construct starters.

It allows a simultaneous consideration of numerous

places in sample genome.

It does not need a prober, radioactive materials, etc.

THE DISADVANTAGES OF RAPD MARKER

The location of RAPD markers is not determined on the

genetic maps.

The similarity and relationship between bands with

similar movement on electrophoresis gel are not deter-

mined.The studies accomplished by Paul et al.(1996)

show that RAPD markers show the genetic distances

between people with little distance from each other

in a less exact way in comparison to other mark-

ers. Therefore, applying markers to taxonomy studies

in species should be considered.These markers have a

dominant power and the disability of allele system in

RAPD markers results in some limitations in the marker

functions.

RANKING HARDSHIP

RAPD has a tendency to propagate repetitive parts of

genomic DNA. As an example, too many repetitive

orders of wheat genome are propagated through RAPD

reaction.

ISSR TECHNIQUE

ISSR is a PCR-based technique contained a piece of DNA

in a reproducible distance between two repetitive  elds

of unique micro-satellites with opposite directs. Usually,

this technique used micro-satellites of 16-25 bp as a

primer of a mono-primary targeting poly-genomic locus

to propagate the successions between micro-satellites

with different measures. Bi-nucleotides, Tri-nucleotides,

quadra-nucleotides, or panta-nucleotides micro-satellite

repetitions can be used as primers. Although applied

markers are connected to 1-4 bases, and are enlarged

based on these connections, they are able to connect to

each point of DNA. This technique has integrated the

advantages of AFLP and micro-satellites with RAPD

comprehensiveness. The applying longer primers (16-25

mers in comparison to RAPD shorter primers (10 mers)

allow to apply high temperature of connection (45-60`C)

increasing the primer connection to determined points

of DNA with more repetitive times results in high repeti-

tion of ISSR. The studies on repetition signify that only

the weakest bands are not repetitive. Nearly 92-95% of

scored pieces can be counted in DNA samples, and can

be repeated in distinct periods of PCR when are identi-

 ed by applying polyacrilamide. 25-50 nanograms of

pattern DNA in each 20 micro-liter of PCR and 10 mil-

ligram of pattern DNA can produce the same propagated

productions. The connection temperature of 45-60`C

depends on the applied primer.

MATERIALS AND METHODS

HERBAL MATERIALS

Nine varieties of wheat species (existed in Minister of

Agricultural Jihad) were applied to consider molecules.

As a whole, nine leaf samples included fresh leaves of

wheat species were collected.Starters applied 14 ISSR

and 10 RAPD starters to consider genetic relationships.

THE EXTRACTION OF GENOMIC DNA

Changed CTAB method of leaf samples was applied to

extract DNA before that samples were melted. To provide

100 milliliter buffer of 2% CTAB extraction, the compo-

nents of table 4-3 were solved in 20 milliliter of distilled

water, pH was 8 by applying chloric acid in one mullar.

Then 2 grams of hexa decyl timthyl ammonium bromide

was solved in hot distilled water, and it was added to the

previous solvent. Finally, the solvent was 100 milliliter.

The phases of DNA extraction are as following:

M. Ebadi and Mahsa Eghbali

290 THE COMPARISON OF ISSR AND RAPD MARKERS WITH DIFFERENT SPECIES OF

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BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Before the experiment was started, CTAB buffer was

heated in water bath of 65`C.

100 milligram of fresh and clean leaves was pounded

in liquid nitrogen in a mortar. It is suggested to keep the

dish and mallet (at least in connection with leaves) cold

by adding some liquid nitrogen or putting in freezer to

make Nucleases inactive.

Put the powder in a corner of the dish, and when

buffer was melted, add 800 micro-liter of CTAB buffer

to powder and mix thoroughly. The contents of mortar

(dish) should be transferred to tube of 2 milliliter, and

was kept in 60`C for 30 minutes in water bath (In this

30 minutes, the tubes were circulated for some times

smoothly). The sample volume (800 micro-liters) of chlo-

roform/ isoamyl alcohol (24:1) was added in room tem-

perature. The tubes were turned some times in a smooth

way to have a uni ed mixture. It was centrifuged for

10 minutes and 13000 turns. The above limpid part was

removed (in 500-600 micro-liters), and was poured in

another tube.

2/3 (almost 350 micro-liters) of cold isopropanol vol-

ume (-20`C) was added to each tube. It was centrifuged

with 13000 turns for 10 minutes in 4`C. the tube con-

tents were removed smoothly and DNA string was left at

the bottom of the tube. Vacant tubes with DNA strings

were inverted on a very clean place to have dry strings.

100 micro-liters of TE buffer was added to room temper-

ature.800 micro-liters of Aminium acetate +cold ethanol

was added (2.5 molar and 1.5 liter of aminium acetate+

3.5 milliliter ethanol), the last step was accomplished on

ice, it was inverted for some times, and then tubes were

transferred to freezer. They were centrifuged with 13000

turns for 15 minutes in 4`C.

The above part was discharged. The tubes were

inverted to become dry. 150 milliliters of TE buffer was

added to each tube. The samples were kept in room tem-

perature, then some other actions to evaluate quantity

(spectro-photometry) and quality (electrophoresis on

agarose gel of 1.5% DNA) (extracted DNA) were accom-

plished. The samples were diluted in 100 nanograms to

subsequent application. Providing method of 0.8% and

1.5% agarose to determine quantity and quality and

analysis of propagated pieces

X1EDTA was diluted by water in 9:1, and then

X1EDTA was provided.

X1EDTA is put in the tube based on the electropho-

resis tank volume by the help of cylinder, it is moved

smoothly to have a good mixture. The solvent in tube

is put in the microwave so that particles will be solved

in a complete way and then a smooth solvent will be

appeared. 0.2 micro-liter of DNA safe stain was added

to gel to paint, then the gel was put in the gel dish. The

solvent in the tube is poured on the electrophoresis tray.

When 30 minutes was passed, agarose gel is stiff. The gel

was put in the electrophoresis tank by X1EDTA.

9-3 sample preparation and electrophoresis of agarose

gel

We add 5 milliliters of loaded buffer to each 5 milliliter

of sample to have a good mixture, and then the sample

is spilled into the sink. The  rst sink from the lift has

been devoted to the measured marker. A 92-volt elec-

tric currency was connected to the tank. By passing 45

minutes, gel was removed from the tank, and then it was

removed from the tray. Then a picture was taken from

the gel in photography machine.

10-3 The components of polymerase chain reaction

The components presented in table 5-3 were applied to

carry out PCR reaction in 12 micro-liter.

Table 4-3. The composed components of DNA extraction buffer

Final destinationAmountBuffer components

100 millimolar1.211 gTris-HCL

4 molar8.19 gNaCl

0.2% (volume-volume)0.744 gEDTA

0.2% (weight-volume)2 gCTAB

0.5 molar200 microliterMerkapto etanol

0.2% (weight-volume)2 gpvp

Table 5-3. The applied components to carry out PCR

reaction

Amount (micro liter)Component

5PCR kate (Master mix)

5Deionized water

1.1Starter

1Genomic DNA

12.1Total

11-3 Time cycle and polymerase chain reaction steps

The polymerase chain reaction was accomplished in

thermocycler (Bio Rad) in 4 minutes and initial com-

pounding in 94`C via 10 initial touching down denatura-

tion (so that the connection temperature of starter was

M. Ebadi and Mahsa Eghbali

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE COMPARISON OF ISSR AND RAPD MARKERS WITH DIFFERENT SPECIES OF

TRITICUM

291

considered to be 5`C higher than the real connection

temperature, and 0.5`C was decreased from the connec-

tion temperature to achieve the real connection temper-

ature). This action allows to decrease similar bands of

micro-satellites causing some problems in scoring the

PCR typical cycles. It is suggested to apply 30 cycles

containing 30 seconds of denaturation in 94`C to each

starter in 45 seconds (to connect starters), and 2 minutes

in 72`C to extend, and the last extension was carried out

in 72`C for 7 minutes.

DATA ANALYSIS

The applied markers in this study are of dominated

markers which are scored in interpreting gel as 1(indi-

cating to the presence) or 0 (indicating to the lack).

CLUSTERING ANALYSIS

According to raw data obtained from ISSR and RADP

molecular markers, the clustering analysis was accom-

plished by UPGMA method and applying the jaccard

similarity coef cient to determine similarity between

two individuals, Darwin 6 and Post 3 software.

THE ANALYSIS TO BASIC COMPONENTS

The analysis to basic components is another multi-vari-

ant method which is of high application in genetic vari-

ety analysis with clustering analysis. This method can

be applied to present two-dimensional distribution of

individuals in a  eld of plot signifying genetic similarity

among them. PCA is a method to decrease data to con-

struct relationships between two or more variants and

to explain the changes of whole basic and primary data

by some new independent variants called basic compo-

nents.To decrease data is accomplished by linear chang-

ing of basic data to new independent variants called

basic components, so that the  rst PC explains the max-

imum initial data, and the second PC explains remained

changes after the  rst PC, etc. it should be noted that

each PC explains those changes not been explained by

other PCs.

As PCs are independent, each one presents differ-

ent properties of basic data, and they should be inter-

fered differently from each other. When PCA is applied

to analyze molecular data, similarity matrix should be

changed via the following formula to remove negative

inert roots:

ij=Sij-Sio-Soj+SooóS

in which, Sij represents similarity coef cient between

I,j individuals, Sio shows the average of similarity coef-

 cient in n

individuals, Soj shows the average of simi-

larity coef cients of j

individual, and Soo is the total

average of similarity coef cients. This changing causes

to move similarity matrix to zero root. These similarity

properties calculated by any method will be kept.

RESULTS AND DISCUSSION

A study accomplished by Aslani (2013) on the genetic

variety of molecular genotype of Mirabilis jalapa by

applying ISSR marker showed that ISSR does not need

radioactive materials and pattern DNA sequence. There-

fore, ISSR was a good marker to consider genetic vari-

ety and relationship which was coincide with our results

(Aslani,2013).

A study accomplished by Heidari Nejad on the

genetic variety of African Violets varieties by apply-

ing RAPD marker signi ed that morphologic properties

are in uence by various factors with no effect on DNA.

These factors which are similar in DNA based on the

data, are similar to or different from each other in mor-

phologic properties. Therefore, it is expected that RAPD

data may have no similarity with individual grouping

in planting and morphological properties by consider-

ing the effectiveness of environmental factors on these

properties.

This result has been reported by Martinz-Gumz et al.

(2003) on almond. Therefore, considering individuals

based on planting and morphological properties can`t

result in favorable results (Heidari Nejad, 2012).

Stephonova et al. (2014) accomplished a study on

genetic variety of 16 species of Amaranthus of Cary-

ophyllales family by applying molecular ISSR marker.

Amaranth is a most important species existing in all

around the world. In this study, ISSR method was applied

to analyze variety in and among 16 species of amaranth.

Eleven primaries were applied in this study. The den-

drogram divided 16 varieties into 3 groups in which 2

groups belong to India and one group is belonged to

Nepal. The similarity average was 0.154-1.000. This

study indicated that ISSR is of high ef ciency (Stepho-

nova et al. 2014).

In a study accomplished by Ray et al. (2007) on the

genetic relationships of Aramanthus which is belonged

to Caryophyllales by applying molecular ISSR and

RAPD markers, it was used from 18 starters and to ISSR

marker and 15 starter to RAPD. The similarity coef cient

of ISSR and RAPD markers were 0.45 and 0.47; respec-

tively. Also, Cophenetic coef cient of both markers was

0.83. These coef cients refer to a good  tting between

similar matrix and dendrogram, and both dendrograms

showed a good similarity between species indicating

that ISSR and RAPD markers are of high suf ciency to

determine genetic relationships, and they are suitable

tools to cluster species (Ray et al. 2007).

292 THE COMPARISON OF ISSR AND RAPD MARKERS WITH DIFFERENT SPECIES OF

TRITICUM

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

M. Ebadi and Mahsa Eghbali

CONCLUSION

In general, the study results signi ed that there is a differ-

ence on the least and most distance of bands in different

analyses. ISSR marker showed bands with less distance

and more similarity in comparison to RAPD marker.

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